American chestnut leaf compositions comprising oxalate oxidase and methods for treatment of oxalate-related disorders

Abstract

The invention provides American chestnut leaf compositions comprising oxalate oxidase. The compositions reduce oxalate levels in dietary sources of oxalate and are useful in reducing oxalate levels and reducing stone disease in patients. The invention provides methods of extracting and purifying oxalate oxidase from American chestnut leaf. The invention provides methods of extracting oxalate from biological samples.

What is claimed is: 
     
         1 . A composition comprising tea leaves and leaves of American chestnut tree plants expressing wheat oxalate oxidase. 
     
     
         2 . The composition of  claim 1 , wherein the leaves of American chestnut tree plants expressing wheat oxalate oxidase are ground. 
     
     
         3 . A method for producing a brewed tea with reduced oxalate levels, comprising:
 (a) combining tea leaves with leaves of American chestnut tree plants expressing wheat oxalate oxidase;   (b) steeping the tea leaves and leaves of American chestnut tree plants expressing wheat oxalate oxidase in boiling water to brew the tea; and   (c) collecting the brewed tea,   wherein the brewed tea has reduced oxalate levels compared to tea brewed without leaves of American chestnut tree plants expressing wheat oxalate oxidase.   
     
     
         4 . The method of  claim 3 , wherein the leaves of American chestnut tree plants expressing wheat oxalate oxidase are ground. 
     
     
         5 . A method of reducing stone formation in a patient, comprising administering a therapeutically effective amount of leaves of American chestnut tree plants expressing wheat oxalate oxidase to the patient, wherein stone formation is reduced in the patient. 
     
     
         6 . A method of reducing urinary oxalate levels in a patient, comprising administering a therapeutically effective amount of leaves of American chestnut tree plants expressing wheat oxalate oxidase to the patient, wherein urinary oxalate levels are reduced in the patient. 
     
     
         7 . A method for purifying oxalate oxidase from leaves of American chestnut tree plants expressing wheat oxalate oxidase, the method comprising:
 preparing an extract from the leaves, wherein the extract is prepared in the presence of 0.1% sodium dodecyl sulfate at 60° C., precipitating proteins in said extract by bringing said extract to at least about 30% (w/v) saturation with ammonium sulfate, removing said precipitated proteins and bringing said extract to at least about 60% (w/v) saturation with ammonium sulfate to precipitate said oxalate oxidase present in said extract and recovering said oxalate oxidase.   
     
     
         8 . An oxalate oxidase composition having a specific activity of at least about 9 enzyme units per 100 g of starting leaves of American chestnut tree plants expressing wheat oxalate oxidase after extraction in the presence of 0.1% sodium dodecyl sulfate at 60° C. and ammonium sulfate precipitation. 
     
     
         9 . The oxalate oxidase composition according to  claim 8  wherein said oxalate oxidase is produced according to a process comprising the steps of preparing an extract from leaves of American chestnut tree plants expressing wheat oxalate oxidase, wherein the extract is prepared in the presence of 0.1% sodium dodecyl sulfate at 60° C., precipitating proteins in said extract by bringing said extract to at least about 30% saturation (w/v) with ammonium sulfate, removing said precipitated proteins and bringing said extract to at least about 60% (w/v) saturation with ammonium sulfate to precipitate said oxalate oxidase present in said extract and recovering said oxalate oxidase. 
     
     
         10 . A method of reducing oxalate levels in a material, comprising adding an effective amount of the oxalate oxidase composition of  claim 8  to the material, wherein the oxalate level of the material is reduced. 
     
     
         11 . The method of  claim 10 , wherein the material is a food or beverage. 
     
     
         12 . The method of  claim 11 , wherein the food or beverage is selected from the group consisting of beer, spinach, rhubarb, okra, peanut butter, taro, bran flakes, soy, tofu, soymilk, nuts, almonds, cashews, peanuts, potatoes, beets, navy beans, raspberries, pumpkin, chocolate, swiss chard, eggplants, yams, avocados, tomato sauce, dates, strawberries, wheat bran, tea, leafy green vegetables, asparagus, runner beans, beetroot, Brussels sprouts, cabbage, carrots, cauliflower, celery, chives, lettuce, marrow, mushrooms, onions, parsley, green peas, radishes, tomatoes, turnips, apples, apricots, ripe bananas, gooseberries, grapefruits, melons, oranges, peaches, pears, pineapples, plums, blueberries, arugula, beet greens, collard greens, kale, endive, bok choy, dandelion greens, escarole, cole, mache, mustard greens, radicchio, rapini, and watercress. 
     
     
         13 . The method of  claim 10 , wherein the material is wood. 
     
     
         14 . A method for extracting oxalate from a biological sample, comprising:
 (a) solubilizing oxalate in the biological sample with 5 M HCl at 60° C. for 4 hours;   (b) separating the solubilized oxalate from insoluble material;   (c) precipitating oxalate from the solubilized oxalate with 12 M ammonium hydroxide;   (d) washing the precipitated oxalate with 5 M ammonium hydroxide; and   (e) resuspending the washed precipitated oxalate in EDTA at a concentration of 0.5 M or 1.0 M.   
     
     
         15 . The method of  claim 14 , wherein the biological sample is tea leaves, leaves of American chestnut tree plants expressing wheat oxalate oxidase, seagrass, spinach, or kidney stone. 
     
     
         16 . The method of  claim 14 , wherein the kidney stone is from a human.